Investigating the contributions of astrocyte gap junctions to ALS disease progression

 描述（由申请人提供）：了解为什么大多数肌萎缩侧索硬化（ALS）患者的疾病进展随时间发生在连续的解剖区域，是设计诊断后可使用的疾病改善疗法的基本限制之一。 来自嵌合体小鼠的研究表明，野生型（WT）和突变型SOD 1星形胶质细胞周围的运动神经元嵌合表达的星形胶质细胞在发病后的疾病传播中发挥作用。然而，星形胶质细胞功能障碍不仅限于转基因ALS啮齿动物模型，而且重要的是，当原位检查以及使用体外人细胞时，星形胶质细胞功能障碍是ALS患者中最一致的观察结果之一。 星形胶质细胞在中枢神经系统（CNS）中通过间隙连接（GJs）和半通道形成高度耦合的细胞间网络，所述半通道由围绕中央孔排列的6个连接蛋白亚基组成。连接蛋白在星形胶质细胞中具有关键作用：稳态缓冲，星形胶质细胞网络的同步化，神经元的代谢支持和血管生理学的调节。它们还可以传播Ca 2+波并调节突触事件或通过半通道释放胶质递质。Cx43是星形胶质细胞中的主要连接蛋白，并且在CNS中普遍表达。 我们的初步数据显示，Cx43在ALS模型以及ALS患者的脊髓和皮质中上调。然而，这种Cx43上调的功能意义尚未在ALS中研究。由于Cx43在星形胶质细胞连接和细胞-细胞信号传导中起作用，我们假设ALS中星形胶质细胞Cx43上调可能导致神经元丢失以及ALS中邻近解剖学疾病的扩散。 该提案将使用啮齿动物来源的星形胶质细胞，重要的是，来自ALS患者的人iPSC来源的星形胶质细胞，在体外解决这些变化的功能意义。还将在体外研究ALS星形胶质细胞中Cx43上调对野生型运动神经元损失的具体影响。最后，我们将利用各种遗传策略以及ALS动物模型中Cx43的局灶性敲除，以解决Cx43的减少是否可以限制疾病传播。这些研究将着眼于将Cx43介导的星形胶质细胞对疾病进展的影响转化为潜在的可能性，并扩展到ALS诊断后干预的潜在药理学策略的发展。

 DESCRIPTION (provided by applicant): Understanding why disease progression in the majority of patients with Amyotrophic Lateral Sclerosis (ALS) occurs in contiguous anatomic regions over time is one of the fundamental limitations to designing disease modifying therapies that can be utilized after a diagnosis. Studies from chimeric mice with mosaic expression of wildtype (WT) and mutant SOD1 astrocytes surrounding motor neurons suggest that astrocytes play a role in disease propagation after onset. However, astrocyte dysfunction is not an observation merely limited to transgenic ALS rodent models but, importantly, has been one of the most consistent observations in humans with ALS when examined in situ as well as using human cells in vitro. Astrocytes form a highly coupled intercellular network in the central nervous system (CNS) through gap junctions (GJs) and hemichannels composed of 6 connexin subunits arranged around a central pore. Connexins in astrocytes have key roles: homeostatic buffering, synchronization of astrocyte networks, metabolic support for neurons, and regulation of vascular physiology. They can also propagate Ca2+ waves and modulate synaptic events or release gliotransmitters through hemichannels. Cx43 is the predominant connexin in astrocytes and is expressed ubiquitously in the CNS. Our preliminary data show that Cx43 is upregulated in ALS models as well as in human spinal cord and cortex in patients with ALS. Yet the functional significance of this Cx43 upregulation has not been studied in ALS. Because Cx43 has a role in astrocyte connectivity and cell-cell signaling, we hypothesize that astrocyte Cx43 upregulation in ALS may contribute to neuronal loss as well as contiguous anatomical disease spread in ALS. This proposal will address the functional significance of these changes in vitro using rodent-derived astrocytes and, importantly, human iPSC-derived astrocytes from ALS patients. The specific influences of Cx43 upregulation in ALS astrocytes on wildtype motor neurons loss will also be investigated in vitro. Finally, we will utilize a variety of genetic strategies as well as focal knockdown of Cx43 in ALS animal models, to address whether reductions in Cx43 can limit disease spread. These studies will occur with an eye towards the potential for translating what is learned about Cx43-mediated astrocytic influences on disease progression and spread to the development of potential pharmacological strategies for intervention after an ALS diagnosis.